The Growth Characteristics in Vitro of Normal Epithelium, Dysplasia, and Carcinoma-//?-^/// of the Uterine Cervix*

RALPH M. RICHART (Departments of Pathology and Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, and the Obstetrical and Gynecological Service [The Sloane Hospital] of the Presbyterian Hospital, New York, N.Y.)

SUMMARY Normal cervical epithelium and epithelium containing only dysplasia and carcinoma- in-situ have been grown in vitro as "pure" cell types without contamination by fibro- blasts or squamous epithelium of a significantly different histological type. Excel lent growth was obtained from all types of tissues explanted, and the morphology and pattern of growth were compared. Individual differences in growth pattern between the different cases of dysplasia and curcinoma-in-situ were noted. These were dis tinctive enough so that the cases could be consistently identified by their morphology without reference to identifying labels. No striking differences in growth rates were noted among the three epithelia, and, although there were remarkable differences in cellular behavior and morphology between the normal and abnormal epithelia, no striking differences were observed between dysplasia and earcinoma-m-sííw.

Numerous authors have reported on the growth of nor saline, a drawing of the distribution of toluidin blue-posi mal cervical epithelium and intraepithelial and invasive tive epithelium is consulted and a biopsy taken, the limits carcinoma in vitro. Considerable variation in success rate of which are contained within the area of iutraepithelial in growing the various types of epithelium has been re neoplasia. This biopsy is placed in a wash solution and ported, and, as well, there has been variability in the taken immediately to the laboratory. It is then split observations made on the outgrowths. The reported longitudinally, one-half retained for histology and the variability may be at least partially accounted for by the other half prepared for tissue culture. A plane is devel difficulty in maintaining rigorous control over the type of oped between the epithelium and the stroma, and the epithelium that is placed in vitro. This paper reports a stroma is stripped away and retained for histological exam series of in vitro observations on normal cervical epithe ination. The stripped epithelium is finely minced, and the lium, dysplasia, and carcinoma-m-stïwin which the type of fragments are placed under perforated cellophane in a epithelium grown in culture was precisely known and special circular micro-glass culture chamber. The cultures rigidly controlled. are incubated in a humidified C02 incubator and are fed routinely 3 times a week. When sufficient outgrowth is MATERIALS AND METHODS obtained the cells are harvested with a solution of 0.25 per The detailed methods used in this study have been de cent trypsin and 0.2 per cent sodium versenate in a bal scribed previously (12) but are summarized in the following anced salt solution, and are replated. paragraphs. According to these procedures 52 biopsies have been Patients are chosen on the basis of abnormal cytologie placed in vitro, including four biopsies of normal epithe examinations based on the differential cell counting tech- lium, eight of carcmoina-in-situ, and 40 of mild, moderate, nic (8, 9). They are examined colpomicroscopically to and severe dysplasia. determine the type of epithelium present on the exposed portion of the cervix (7), after which the distribution of RESULTS toluidin blue-positive epithelium is noted (10), and, if the Fifteen of the biopsies were grown in Puck's F4-FC area of intraepithelial neoplasia is large enough so that one media and 37 in Eagle's minimal essential media with 15 or more biopsies may be taken that will include only blue- per cent fetal calf serum. Growth was obtained in all the staining epithelium, the patient is chosen for study. At biopsies in Puck's media, but it was impossible to maintain the next visit the cervix is thoroughly washed with sterile the culture or to sub-culture by this media. The discus sion of results will be confined to the 37 biopsies which «Aided by USPHS grants: C-9800, GM-K3-13975, RG-8165, were grown with Eagle's. In 27 of these biopsies excellent and a General Research Support Grant from the National Insti tutes of Health. growth was obtained. In 22 the outgrowth was composed Received for publication September 27, 1963. of epithelial cells alone, whereas in five there was fibro- 002

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1964 American Association for Cancer Research. RicHART—Carcinoma-m-siiu. of Uterine Cervix 663 blastic contamination. One culture was contaminated by DISCUSSION fungi within the first 3 days after culture, and one of the In a recent paper Mellgren et al. (3) studied the growth plates containing good growth became contaminated after rate of normal epithelium, dysplasia, and carcinoma-in-situ several weeks in culture. In nine biopsies there was no as well as invasive carcinoma. They found that, although growth, but two of these occurred as a result of an incuba invasive carcinoma had a higher mean growth rate than tor accident. did other forms of cervical epithelium, there was no differ The first outgrowth was observed in all types of epithe ence between the growth rate of dysplasia or carcinoma-m- lium between the 4th and 7th days, the great majority situ and normal epithelium from the same patients. This occurring at 6 days after explantation. The normal epithe observation is in keeping with our findings. Moore, in lium characteristically grew out as a confluent monolayer two papers (4, 5), has described growth characteristics of composed of a mosaic of uniform cells (Figs. 1, 2, 3). The normal cervical epithelium, carcinoma-zn-si/M and invasive most striking feature of the outgrowth was that it was a carcinoma with roller tubes and the Maximow double confluent sheet, each cell border being intimately juxta coverslip technic. He has described one case of carci- posed to the neighboring cells. The only place a free noma-in-situ which was cultured on two different occasions border was found was at the growing edge of the sheet. with good growth each time, in which the cell type was Mitoses were frequent, and the growth, as estimated from said to be of a uniform pattern, well differentiated with an the number of mitotic figures observed, was sustained at a extensive, regular mosaic of cells. This description sug relatively constant rate which did not appear to differ strikingly from that of dysplasia or carcinoma-m-siiw. gests that the observations were made on portions of nor mal epithelium, and Moore has alluded to this possibility After approximately 1 week in culture, scattered areas in by stating that the tissue placed in culture "may not in the normal epithelium began to change in their morphology clude a sample of the specific area desired for study." and to be composed of larger and flatter cells with small Grand (2) noted differences between normal epithelium, centrally placed nuclei (Fig. 4). These cells were not un dysplasia, carcinoma-in-s¿¿w,and invasive carcinoma, like the cells of superficial cervical epithelium, and in the particularly related to the time after explantation in which areas in which this change occurred no further mitotic activity was noted. outgrowth occurred and in the growth rate. He reported In the dysplasia and carcinoma-m-s#u group virtually that the time for outgrowth to occur decreased progres sively and the growth rate increased progressively as one every case had a separate and distinctive morphology (Figs. 5-13). The differences in growth pattern resulted passed from normal epithelium through dysplasia and carcinoma-m-sîiwto invasive carcinoma. This is at primarily from variations in the degree of cell cohesion, variance with our results. The author does not state how differences in the numbers of cell processes which were the biopsy sites were chosen, nor are there any photographs extended, and differences in propensity toward migration. illustrating either the types of epithelium placed in vitro or In the latter instance cells in the cultures maintained the pattern of the growth. Zinser (13) in cover-glass different degrees of dispersion at the periphery of the cultures compared normal cervical epithelium with inva colonies, and in some cases isolated cells migrated for con sive carcinoma and carcinoma-m-stÕMand found that siderable distances away from the main areas of growth. differences in growth activities were appreciable only be It has not been possible to predict the pattern of growth tween normal and abnormal epithelium on the one side and thefrom dysplasias the histology and of carcinomas-in-sz'fw the explanted epithelium. exhibit a Most striking of carcinoma and carcinoma-¿n-s¿íwonthe other. Glatthaar (1) could find no growth whatsoever in normal epithelium degree of lack of cell contact which results in a more dis or "agitated and abnormal epithelium," but says this persed culture in which numerous cell processes are seen, tissue could be kept alive and could be studied by phase- giving these cells an irregular outline. contrast microscopy and stated, "the cells are the very Because of the unique morphology of each individual same we find in vaginal smears." culture one quickly learns the morphological characteristics It seems to be agreed that normal cervical epithelium is of each and can distinguish among a small group of them technically more difficult to grow in vitro than are the ab consistently, without referring to identifying numbers on normal epithelia. Although Glatthaar was unable to the culture plate, by noting the growth pattern and cellular grow normal epithelium and Moore and Grand both men morphology. Of the 22 cultures in which good epithelial growth un- tioned the difficulty in obtaining growth, it appears from contaminated by fibroblasts was obtained, all were success our studies and from those of Mellgren (3) and Mulligan fully transferred for one or more transfer generations, and (6) that it is possible to obtain excellent growth and that many have been repeatedly frozen and thawed and re- the cells may be maintained in vitro for long periods of time and even transferred. In our experience the major plated. The distinctive morphology of the dysplasias and carcinomas-m-stÕMis usually maintained through at difficulty in establishing normal epithelium in vitro is in obtaining the proliferative layer uncontaminated by least two transfer generations and frequently for more, but cervical stroma. In a radioautographic study of cervical the cultures of normal epithelium that are successfully epithelium (12) it was shown that the proliferative cells transferred become much less regular after transfer, losing occur primarily in the basal and parabasal cellular layers the pavement mosaic type of growth and appearing similar and, because of the adhesiveness of normal epithelium to to the neoplastic epithelium. The "maturation" was not its subjacent stroma, these areas may be extremely difficult observed in the transfer generations of normal epithelium. to remove. Most of our unsuccessful cultures of normal

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1964 American Association for Cancer Research. 664 Cancer Research Vol. 24, May 1964 epithelium were composed primarily of superficial and By examining the patient with a colpomicroscope prior to intermediate cells which were at a mitotic end stage (11) biopsy to determine that the entire field to be biopsied is and incapable of growth in vitro. composed of the type of epithelium desired, and by saving The results of a variety of authors' studies of cervical one-half of the biopsy for histological examination, it can epithelium in vitro, particularly the more recent studies be determined with a high degree of certainty that the type utilizing more adequate media than was available previ of epithelium being placed in vitro is of essentially one ously, appear to indicate that the major difference in vitro morphological type uncontaminated by any other type. between normal cervical epithelium and cervical intraepi- This can be confirmed by noting that the outgrowth is thelial neoplasia (dysplasia and carcinoma-m-stïw)is one composed only of epithelial type cells and that the mor of morphology. Normal epithelium grows in a pavement- phology is distinct from plate to plate. Utilizing this like mosaic with intimate cell contact and a growth rate material it should be possible to more clearly define the which is similar to that of intraepithelial neoplasia. Dys differences between normal cervical epithelium and plasia and carcinoma-m-«Ywdifferfrom normal epithelium cervical intraepithelial neoplasia. primarily by the lack of cell contact migratory capacity and variable cellular morphology, but there are no striking REFERENCES 1. GLATTHAAB,E. C. Superficial Carcinoma of the Cervix Uteri; differences in growth rates as determined by relatively Its Possible Differentiation from Atypical Epithelium by crude technics. Significantly, no differences have been Use of Tissue Culture. Am. J. Obst. Gynecol., 61A:(Suppl.) noted consistently between the various grades of intraepi 73-82, 1951. thelial neoplasia, and it has been impossible to consistently 2. GKAND, C. G. Cytologie-Tissue Culture Studies on Cervical Epithelium. Ann. N.Y. Acad. Sci., 63:1436-40, 1956. predict from the growth in vitro the histological appear 3. MELLGREN, J.; BOEBYD, B.; AND RAGMAN, M. Growth Po ance of the epithelium from which the culture was derived. tentials of Precancer of the Cervix Uteri in Vitro and in Corti The fact that we have observed striking and character sone-treated Hamsters. Cancer Res., 22:13ÕM6,1962. istic differences which are relatively unique from patient 4. MOOHE, J. G. Growth Characteristics of Normal and Malig nant Cervical Epithelium in Tissue Culture. Am. K. Obst. to patient in the tissue placed in vitro, while other authors Gynecol., 64:13-24,1952. have not made this observation, may probably be attrib 5. . Growth Characteristics in Tissue Culture of Con uted to the fact that patients are chosen for study only if troversial Lesions of the Uterine Cervix. Western J. Surg. the lesion is of such a size and distribution that a biopsy 63: 1-9, 1955. may be taken which will contain only epithelium of a 6. MULLIGAN, R. M. Growth of Human Cervical Epithelium in homogeneous and uniform type—i.e., either normal Tissue Culture. Tissue Culture Assoc. Abstracts, 13th An nual Meeting, 1962. epithelium, dysplasia, or carcinoma-m-sííu.This epithe 7. OKAGAKI, T.; AUSTIN, J. H.; AND YOUNGE, P. A. Colpomi- lium is sampled with a Kevorkian punch, an instrument croscopy of Anaplasia (Dysplasia) and Carcinoma-ín-stíwof that is specifically designed for cervical biopsies, and the Uterine Cervix. Acta Cytol., 6:403-4, 1962. removes a small, oblong piece of tissue with little subja 8. OKAGAKI, T.; LERCH, V.; YOUNGE, P. A.; McKAY, D. G.; AND KERVORKIAN, A. Y. Diagnosis of Anaplasia and Car- cent stroma which is easy to manipulate for tissue culture studies. Mellgren states that "the carcinoma-m-stiu cinoma-in-stitt by Differential Cell Counts. Acta Cytol. 6:343- 47, 1962. epithelium in vitro closely resembled the normal epithe 9. RICHART,R. M. Cervical Neoplasia in Pregnancy: A Series of lium, although that from occasional patients exhibited a Pregnant and Post-Partum Patients Followed without Biopsy characteristic pointed cell form and slight pleomorphia." or Therapy. Am. J. Obst. Gynecol., 87:474-77, 1963. He also states that the lesions often occupy only a "small 10. . A Clinical Staining Test for the in vivo Delineation of Dysplasia and Carcinoma-in-sti«. Ibid., 86:703-12, 1963. fraction of the sample." This source of error was also 11. . A Radioautographic Analysis of Cellular Proliferation recognized by Moore, as mentioned previously. Through in Dysplasia and Carcinoma-ira-sziw of the Uterine Cervix. the use of the toluidin blue stain and the Kevorkian punch Ibid., pp. 925-30, 1963. we have been able to obtain "pure" normal or neoplastic 12. . A Method for the Growth of "Pure" Cervical Epi thelium in Vitro. Ibid, (in press). epithelium, and by tediously stripping epithelium from the 13. ZINSER, H. K. Tissue Culture and Carcinoma-ira-sz'íií.Acta stroma there is little or no fibroblastic contamination. Cytol., 6:159-60, 1962.

FIG. 1.—Photomicrograph of normal epithelium from which cultures illustrated in Figures 3 and 4 were derived. Hematoxylin and eosin (H. & E.), X220. FIG. 2.—Culture of normal epithelium. Note the homogeneous appearance of the confluent pavement-like monolayer. There is one metaphase figure at upper center and a prophase at left cen ter. Phase-contrast, X260. FIG. 3.—Culture of normal epithelium. The pattern of growth is similar to that described in Figure 2. There are three metaphase and one telophase figures in the field. At the lower left the active epithelium is seen to merge with the areas that are beginning to "differentiate." These cells are of a lighter hue, owing to their being flatter and having less cytoplasmic activity. Phase-con trast, X260. FIG. 4.—Culture of normal epithelium. An area of "differenti ation." The cells are flattened, many of the nuclei are smaller than the nondifferentiated epithelium, cytoplasmic activity is decreased, and no mitotic activity is observed. Phase-contrast, X260.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1964 American Association for Cancer Research. FIG. 5.—Photomicrograph of biopsy from which cells in Figure 6 were derived. Interpreted as moderate dysplasia. H. & K., X220. Fio. ti.—Culture of moderate dysplasia. Although the cells are closely packed, they still tend to grow as single cells and not as a confluent sheet. Although there are cell-cell contacts they rarely extend along an entire side of the angular cells. There is no pleomorphism, but occasional binucleate cells are seen. Phase- contrast, X260. FIG. 7.—Photomicrograph of biopsy from which culture in Fig ure 8 was derived. Interpreted as mild dysplasia. H. & E., X220. FIG. 8.—Cultureof mild dysplasia. Most of the cells are polyg onal forms, but some tend to be elongate. There is striking lack of cell contact. Refractile cells in the upper left portion of the field are in mitosis. Phase-contrast, X 260.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1964 American Association for Cancer Research. FIG. 9.—Photomicrograph of biopsy from which culture in Figures 1(1und 11 was derived. Interpreted as moderate dysplasia. H. & E., X22Ãœ. FIG. 10.—Culture of moderate dysplasia. Even in this area of high population density in which the cells are juxtaposed because of crowding, the cell borders are distinct. '1he cells cither grow adjacent to one another with little contact or overlap one an other. Several mitoses are evident. Phase-contrast, X2GO. FIG. 11.—Peripheral area of culture illustrated in Figure 10. The degree to which the cells grow separately is accentuated at the margins of the colonies. Although some cell-cell contact is evident, it is never of striking degree. There is one metaphase figure in the center of the field. Phase-contrast, X320. FIG. 12.—Photomicrograph of biopsy from which cells in Fig ure 13 were derived. Interpreted as carcinoma-in-sz'ÕM. H. &.E., X220. FIG. 13.—Culture of carcinoma-in-s¿<¡/. The cells and growth pattern are not unlike those seen in the dsyplasias. There is no pleomorphism. The cells tend to grow separately with only a small degree of contact. 1 here is a mitosis nearing completion in the center of the field. Phase-contrast, X320.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1964 American Association for Cancer Research. The Growth Characteristics in Vitro of Normal Epithelium, Dysplasia, and Carcinoma- in-situ of the Uterine Cervix

Ralph M. Richart

Cancer Res 1964;24:662-669.

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